Heriot-Watt University Research Gateway

Heriot-Watt University

Influence of temperature, needle gauge and injection rate on the size distribution,concentration and acoustic responses of ultrasound contrast agents at high frequencySun, Chao; Panagakou, Ioanna; Sboros, Vassilis; Butler, Mairead Benadette; Kenwright,David; Thomson, Adrian J. W.; Moran, Carmel M.Published in:Ultrasonics

DOI:10.1016/j.ultras.2016.04.013

Publication date:2016

Document VersionPeer reviewed version

Link to publication in Heriot-Watt University Research Portal

Citation for published version (APA):Sun, C., Panagakou, I., Sboros, V., Butler, M. B., Kenwright, D., Thomson, A. J. W., & Moran, C. M. (2016).Influence of temperature, needle gauge and injection rate on the size distribution, concentration and acousticresponses of ultrasound contrast agents at high frequency. Ultrasonics, 70, 84–91. DOI:10.1016/j.ultras.2016.04.013

General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Download date: 16. May. 2018

Accepted Manuscript

Influence of temperature, needle gauge and injection rate on the size distribution,

concentration and acoustic responses of ultrasound contrast agents at high fre-

quency

Chao Sun, Ioanna Panagakou, Vassilis Sboros, Mairead B. Butler, David

Kenwright, Adrian J.W. Thomson, Carmel M. Moran

PII: S0041-624X(16)30024-5

DOI: http://dx.doi.org/10.1016/j.ultras.2016.04.013

Reference: ULTRAS 5262

To appear in: Ultrasonics

Received Date: 18 September 2015

Revised Date: 16 February 2016

Accepted Date: 16 April 2016

Please cite this article as: C. Sun, I. Panagakou, V. Sboros, M.B. Butler, D. Kenwright, A.J.W. Thomson, C.M.

Moran, Influence of temperature, needle gauge and injection rate on the size distribution, concentration and acoustic

responses of ultrasound contrast agents at high frequency, Ultrasonics (2016), doi: http://dx.doi.org/10.1016/j.ultras.

2016.04.013

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Influence of temperature, needle gauge and injection rate on the size

distribution, concentration and acoustic responses of ultrasound

contrast agents at high frequency

Chao Sun, *$ Ioanna Panagakou, * Vassilis Sboros, *† Mairead B Butler, *† David

Kenwright, * Adrian JW Thomson, * Carmel M Moran *

*Medical Physics, Centre for Cardiovascular Research, University of Edinburgh,

Edinburgh, UK; $Ultrasound Department, Xijing Hospital, Xi’an, China; †Institute of

Biochemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh,

UK

Corresponding author: Carmel M Moran

Email: carmel.moran@ed.ac.uk

Postal address:

Room E3.06, Centre for Cardiovascular Science, Queen's Medical Research Institute

The University of Edinburgh

47 Little France Crescent

Edinburgh, UK

EH16 4TJ

2

ABSTRACT

This paper investigated the influence of needle gauge (19G and 27G), injection rate

(0.85ml·min-1, 3ml·min-1) and temperature (room temperature (RT) and body

temperature (BT)) on the mean diameter, concentration, acoustic attenuation, contrast

to tissue ratio (CTR) and normalised subharmonic intensity (NSI) of three ultrasound

contrast agents (UCAs): Definity, SonoVue and MicroMarker (untargeted). A

broadband substitution technique was used to acquire the acoustic properties over the

frequency range 17–31 MHz with a preclinical ultrasound scanner Vevo770

(Visualsonics, Canada). Significant differences (P<0.001 - P<0.05) between typical in

vitro setting (19G needle, 3ml·min-1 at RT) and typical in vivo setting (27G needle,

0.85ml·min-1 at BT) were found for SonoVue and MicroMarker. Moreover we found

that the mean volume-based diameter and concentration of both SonoVue and

Definity reduced significantly when changing from typical in vitro to in vivo

experimental set-ups, while those for MicroMarker did not significantly change. From

our limited measurements of Definity, we found no significant change in attenuation,

CTR and NSI with needle gauge. For SonoVue, all the measured acoustic properties

(attenuation, CTR and NSI) reduced significantly when changing from typical in vitro

to in vivo experimental conditions, while for MicroMarker, only the NSI reduced,

with attenuation and CTR increasing significantly. These differences suggest that

changes in physical compression and temperature are likely to alter the shell structure

of the UCAs resulting in measureable and significant changes in the physical and high

frequency acoustical properties of the contrast agents under typical in vitro and

preclinical in vivo experimental conditions.

KEY WORDS: High frequency ultrasound, microbubble, needle gauge, injection rate,

temperature, acoustic characterisation

3

1 INTRODUCTION

There is an increasing number of applications for the use of UCAs in the preclinical

field [1], with an associated increase in the number of published studies utilising

UCAs both in vivo and in vitro [2-5]. However, the differences between the

administration of UCAs in an in vivo environment at body temperature and an in vitro

experiment at room temperature need to be considered. The size of needle gauges

used for small animal in vivo injections are often not cited in the literature but

generally range from 24G for rat tail vein injection [6] , 27G for mouse tail vein

injection [7] and in larger animals - 30G for pig cervical region of nerve [8] and dog

lymphatic system [9]. Generally in vitro experiments of UCAs are performed at room

temperature using 18G - 20G needle gauges – sizes which are widely used in clinical

practice and specifically recommended by some contrast agent manufacturers [10].

When bolus injections are undertaken in small animals, although the bolus is

considerably smaller (of the order of microliters), it is generally performed at an

injection rate of between 0.5 - 3 ml/min, a rate that is commonly used for clinical

bolus intravenous injections.

1.1 The influence of temperature

The three commercial UCAs: Definity (Lantheus Medical Imaging, USA), SonoVue

(Bracco, Italy) and MicroMarker (untargeted) (Visualsonics, Canada) are lipid-coated

microbubbles (MBs). The shells of the three UCAs are monolayers but each is formed

from a varying combination of different lipids. As a result, it is difficult to determine

a specific phase transition temperature for the lipid shells of each and hence the effect

of temperature on the lipid-coated MBs is unknown and its resultant influence on

microbubble characteristics is unclear. The fluid-gel phase transition temperature of

4

lipids in bilayer states varies from -1°C to 75°C [11] and -1°C to 55°C [12] depending

on the phospholipids acyl chain length. Lipid molecules are limited on the membrane

plane in two phases – either fluid: liquid phase; or gel: solid phase, where the liquid

phase allows a freer diffusion of molecules than in the solid phase [13]. For this

reason, the viscosity, elasticity, free energy and diffusion coefficient of the MB lipid

shell can be changed below, at, or above the transition temperature [12]. The lipid

shell of Definity consists of three phospholipids (DPPA, DPPC and MPEG 5000

DPPE) [3]. The lipids incorporated into MicroMarker shell are polyethylene glycol,

phospholipids (unspecified) and fatty acids (VisualSonics PN11691) [14]. SonoVue

possesses an amphiphilic phospholipid shell (a hydrophilic surface outside and a

hydrophobic surface inside) [15] and involves two lipids (DSPC and DPPG) [16].

The thermal response of SonoVue has previously been studied over the temperature

range 37-43oC and its lipid transition temperature was inferred to be 40oC, from

observation of changes in its maximal diameter and backscatter intensity (ultrasound

frequency: 2.5- 8MHz, low MI: 0.0081- 0.113) [17]. For Definity and MicroMarker,

the attenuation was measured and found to be higher at 37°C than at 25°C over a

frequency range from 2 to 25MHz. Measurements were undertaken in bovine serum

albumin and whole blood and no difference in attenuation was found between the two

diluents [14]. Individual Definity and SonoVue MBs have previously been measured

at room temperature (21oC) and body temperature (37oC) using a high speed optical

camera under an insonation of 1.7MHz, 10-80kPa pulse [18]. Compared with the

performance at room temperature, Definity and SonoVue showed an increase in radial

expansion and a decrease in onset of acoustic oscillation at body temperature.

Extensive temperature dependence studies of SonoVue were completed using 3.5

5

MHz-ultrasound [16, 19]. Increasing the temperature close to the transition

temperature resulted in bubbles exhibiting greater radial expansion. Expanding MBs

and rapid diffusion was shown to increase the mean diameter, attenuation, backscatter

and nonlinear components.

1.2 The effect of needle size and injection rate

Talu [20] measured the variation in concentration and mean diameter of one in-house,

lipid-encapsulated UCA at 3 concentrations (1, 5, 10×108 MBs·ml-1) using 3 needle

gauges (23G, 27G and 30G) at 5 injection rates (0.01, 0.03, 0.1, 0.3 and 0.5 ml.sec-1).

With increasing needle gauge (decreasing inner needle diameter), the measured

concentration of microbubbles was shown to decrease suggesting destruction of the

MBs. In addition, a reduction in mean diameter was observed and attributed to an

overall decrease in number of MBs in the population. It was suggested that this was

due to diffusion by the forced compression and possible preferential destruction of

large MBs. A similar study investigating the influence of administration variables (2

needle gauges: 18G and 25G; 2 syringes: 5ml and 10ml; 5 discrete flow rates from 2

to 3.3 ml·min-1; 2 suspending fluids: distilled water and 95% volume-glycerol) was

performed using another in-house lipid MB [21]. The results from this study showed

that in addition to the hydrostatic pressure, shear stress due to the increasing pressure

and velocity gradient played a key role in destroying MBs. However, this study found

that an increasing volume flow rate (i.e., injection rate) reduced the destruction of

MBs, which is not in agreement with the study of Talu [20]. The discrepancy was

attributed to the difference in initial diameter, size distribution, concentration and

composition of MBs. An in vivo experiment to improve plasmid transfection using

SonoVue MBs-mediated gene transfection tested the effects of using 3 needle gauges

6

(25G, 27G and 29G) and showed that increasing needle gauge increased MB

destruction and reduced MB diameter [22].

From the above studies, temperature, needle gauge and injection rate have been

shown to have significant effects on the size distribution and acoustic properties of

UCAs. However, the implications of these results for preclinical studies remain

unclear. This is due to several factors. Firstly, the previous studies are limited to the

ultrasound frequencies relevant for clinical applications and little research has been

performed at high frequencies applicable for preclinical applications. Secondly, the

effect of temperature, needle gauge and injection rate have been discussed separately

but the potential interactions between them remain indistinct. Thirdly, a wide range of

in-house MBs have been studied while commercial UCAs (both clinical and

preclinical) are the products that are most commonly used for preclinical studies and

the influence of these parameters on these commercial agents has not been fully

investigated at high frequencies.

The aim of this paper is to investigate the influence of needle gauge, injection rate and

temperature on the mean diameter, concentration, acoustic attenuation, contrast to

tissue ratio (CTR) and normalised subharmonic intensity (NSI) of solutions of

Definity, SonoVue and MicroMarker (untargeted) over the frequency range of 17-

31MHz. In particular, the changes observed in these measurements for SonoVue and

MicroMarker, the most commonly used contrast agents for preclinical studies, under

typical in vivo and in vitro experimental conditions will be discussed.

2 METHOD AND MATERIALS

7

2.1 UCAs preparation

UCAs were reconstituted based on the manufacturers’ guidelines and were ready for

experimental use after standing at room temperature for 20 minutes. The information

of the three UCAs is listed in Table 1. Based on the maximum concentration from the

manufacturer’s published literature, MBs were diluted in air saturated distilled water

to reach a concentration of 0.8×106 MBs·ml-1. At this concentration, the occurrence

of multiple scattering and shadowing of Definity MBs was previously shown to be

avoided [23].

8

Table 1: The parameters and dilution of contrast agents, *Definity [10], SonoVue [24, 25], ‡MicroMarker [26]

Name Gas Shell Number-

based mean

diameter

(µm)

Maximum

concentration after

reconstitution

(mbs/ ml)

Dilution

in this

study

Manufacturer

* Definity® C3F8 Phospholipids 1.1-3.3 1.2×1010 1:15000 Lantheus Medical

Imaging, USA

†SonoVue® SF6 Phospholipids 2-3 5×108 1:625 Bracco, Italy

‡MicroMarkerTM

untargeted contrast

agent

C4F10 /

N2

Polyethylene glycol,

Phospholipids and fatty

acid

2.3-2.9 2×109

1:2500 Visualsonics, Canada

9

2.2 The control of needle gauge and injection rate

In this study, two needles (19G and 27G) (Becton, Dickinson and Company (BD),

USA) are selected: a 19G (internal diameter (ID) = 0.686mm) needle is generally used

in human intravenous injection and also in some in vitro experiments while a 27G (ID

= 0.21mm) needle is commonly used for mouse-tail injections and mouse intra-

cardiac injections. Two injection rates are studied: 0.85 ml·min-1 and 3ml·min-1, where

0.85 ml·min-1 is a typical injection rate used in bolus injections into a mouse tail vein

during preclinical studies and 3 ml·min-1 a typical injection rate applied in in vitro

experiments. The steady and reproducible injection rate was controlled using a

syringe pump (Aladdin, World Precision Instruments Inc., USA) by connecting the

test needle with 1ml-syringe (ID = 4.78 mm) to reach an injection rate of 0.85 ml⋅min-

1 and 2ml-syringe (ID=8.66 mm) to reach a 3ml·min-1injection rate.

2.3 Temperature control and the measurement of dissolved oxygen level

The entire experiment was undertaken in air saturated distilled water (diluent) at both

room temperature (RT: 20 2oC) and body temperature (BT: 37 2oC). Two-degree

Celsius is the maximal range of variation in temperature. The water was determined to

be air-saturated using the dissolved oxygen content in the water. This was monitored

using a dissolved oxygen probe (Vernier Software & Technology, OR, USA) using

the assumption that oxygen (O2) and nitrogen (N2) are the dominant gases in the water

and that the dissolved O2 is proportional to the N2 at atmospheric pressure under

Henry’s law [27]. The air-saturated water at RT was prepared by leaving the distilled

water over 24 hours. The air-saturated water at BT was obtained by heating the water

to 42oC then degassing using a vacuum pump. The resultant air saturated water at BT

± ±

10

was sealed in a bottle and placed on a hotplate, to maintain the temperature of the

water throughout the experiment.

For the process of withdrawing Definity and MicroMarker, a 19G needle was inserted

into the vial of activated contrast agent, a second 19G was inserted for venting the gas

as per the manufacturer’s recommendations. One of the 19G needles was connected to

a syringe and the Definity or MicroMarker was withdrawn at a steady slow rate of

approximately 1.5ml⋅min-1. For SonoVue, once it was formulated it was withdrawn

from its vial using a needleless syringe provided by the manufacturer at a speed

similar to that used to withdraw Definity and MicroMarker. The choice of syringe

selected for the experiments depended on the injection rate selected (1ml-syringe for

0.85 ml·min-1 and 2ml-syringe for 3ml·min-1). The syringe was then placed

horizontally on a syringe pump and connected with one of the needles under test. At

the injection rate setting, UCAs were collected from the needle tip into an Eppendorf

PCR tube and were then diluted ready for sizing and acoustical measurement. The

experiments undertaken at BT were performed in an identical manner except that the

diluent was the prepared air-saturated water at BT. Because the MBs suspension was

placed in an open tank and stirred continuously throughout the acoustic measurements,

the water tank was placed on a hotplate to maintain the temperature at BT.

Details of the different combinations of needle gauge, injection rate and temperature

for each experiment are shown in Table 2. In this study, the choice of a 19G needle,

an injection rate of 3ml·min-1 in a diluent at RT is defined as ‘in vitro’ representing a

typical in vitro situation. The choice of a 27G needle, an injection rate of 0.85ml·min-1

in a diluent at BT is defined as an ‘in vivo’ experimental setting. For analysis of the

11

data, the ‘in vitro’ setting was defined as the control group and all data was compared

to this. Consequently for each agent, comparison between the 19G and 27G

experimental data at the same injection rate (3ml·min-1) and temperature (RT) reveals

impact of the needle gauge. Comparison between the 3ml·min-1 and 0.85ml·min-1 at

the same needle gauge (19G) and temperature (RT) enables the impact of the injection

rate to be determined, and between the RT and BT at the same needle (19G) and

injection rate (3ml·min-1) the impact of temperature can be determined for

MicroMarker and SonoVue. Finally between the in vitro and in vivo settings the

combined impact of temperature, injection rate and needle gauge size can be

determined. For Definity and SonoVue, the mean diameter was acquired for all

experimental settings, however for MicroMarker only the data from the in vitro and in

vivo were acquired. The acoustical properties of Definity at 370C are not included in

this study.

12

Table 2. The specific experimental settings for the five groups

2.4 Characterisation of diameter and concentration

The mean diameter of the three UCAs was measured by a laser diffraction particle

analyser Mastersizer 2000 Hydro MU (Malvern Instruments Ltd, Malvern, UK).

Three measurements were taken from each sample and the mean values displayed.

The variation in concentration was measured using haemocytometers (FastRead 102

Counting Slides, Immune Systems Ltd, UK) and an inverted microscope (Zeiss

Axiovert 25) under a 40× magnification. MBs were manually counted in a 4×4-

counting grid for each experimental set-up. Each experimental setting was repeated

three times for each agent yielding a mean value of 9 counts for each agent at each

experimental setting.

2.5 Acoustic characterisation

A Vevo 770 preclinical ultrasound scanner (VisualSonics Inc., Canada) was used to

measure the acoustic properties of the contrast agents. Details of the measurement

technique can be found in [28, 29] and only a brief summary will be described here.

The transducer was placed perpendicular to a polymethylpentene (TPX) reflector

Group name In vitro Needle Gauge Injection

Rate

Temperature In vivo

Needle gauge 19G 27G 19G 19G 27G

Injection rate

(ml·min-1)

3 3 0.85 3 0.85

Temperature RT RT RT BT BT

13

(Boedeker Plastics, Shiner, TX, USA) in a water tank (length: width: height = 4cm: 1

3cm: 5cm) on a magnetic stirrer (RCT basic, IKA, US). A magnetic bar (3mm-OD, 2

1cm-length) was placed in the tank to ensure a homogeneous MB suspension. One 3

transducer 707B with nominal centre frequency of 30MHz, a focal length of 12.7mm, 4

a 3dB bandwidth ranging from 17-31MHz, a 6dB bandwidth of 13-35MHz at 3% 5

transmitting power (the peak negative pressure (PNP) was measured to be -0.56 MPa) 6

performed as both a transmitter and receiver. The PNP was confirmed using a 7

membrane hydrophone with a 0.2mm diameter active element made of Polyvinylidene 8

Fluoride (PVDF) (Precision Acoustics Ltd., Dorchester, UK). The PNP of -0.56 MPa 9

was chosen as we have previously demonstrated that microbubbles were not disrupted 10

at this PNP and frequency at RT [29]. 11

12

2.6 Data analysis 13

The attenuation and contrast to tissue ratio (CTR) of the MB suspension were 14

measured in this study to evaluate the fundamental acoustic signature of UCAs. The 15

measurements of attenuation coefficient, α, and CTR as a function of frequency are 16

described in more detail in Sun et al. [29]. In this study, both the attenuation and CTR 17

were averaged over the 3dB bandwidth of the transducer giving a mean value. 18

19

The normalised subharmonic intensity (NSI) of backscatter is measured to 20

characterise the nonlinear property of the UCAs using Equation 1. 21

(Eq.1) 22

14

where the numerator is the subharmonic component of the mean backscattered signal

measured over a 2MHz bandwidth centred over the subharmonic frequency (15MHz)

and the denominator is the mean backscattered signal measured over a 2MHz

bandwidth over the fundamental frequency (30MHz) of the in vitro case (i.e., 19G

needle, 3ml·min-1 at RT).

The data analysis used the radio-frequency (RF) data acquired from a pre-selected

region of interest (ROI) (1.05 mm × 1.6 mm) centred over the focal position of the

transducer. The RF data was output and calculations were performed offline using

MATLAB software (MATLAB 2009A, The MathWorks Inc., Natick, MA, USA).

Each experiment was repeated three times and 900 independent samples (300

consecutive frames on 3 lines) in each ROI were collected per experiment. The error

bars were the standard deviation calculated from three independent measurements.

For the measurement of attenuation and CTR, a single cycle signal was used as the

driving pulse. For the measurement of NSI, modified VisualSonics software was used

to generate a 25-cycle pulse (3% power, PNP equal to -0.67 MPa) and the

subharmonic response was isolated from the receiving signals.

2.7 Statistics

A student’s T test was performed between the in vitro case and all other combinations

of needle gauge size, injection rate and temperature to investigate whether there is a

significant difference in the mean diameter and acoustic properties of UCAs in the

frequency range 17-31MHz due to the variation in the needle gauges, injection rate

and temperature. The significance level was set at 0.05. The results of the student’s T

15

test are marked on the top of each group using the in vitro result as control in Figures

1 - 5.

2.8 Pressure drop and shear stress in the syringes and needles

The pressure drop from the syringe to the needle and the shear stress, τ, in the

syringes and needle were calculated using the equations found in [21].

3 RESULTS

3.1 Pressure drop and shear stress in the syringes and needles

Table 3 lists the calculated shear stress in the syringes and needles and the pressure

drop from the piston in the syringe to the outlet of the needle at different settings. At

certain injection rates (0.85 ml/min using 1ml-syringe, 3ml/min using 2-ml syringe),

the pressure drop and the shear stress in the needle increase with increasing needle

gauge (i.e., decreasing I.D.).

16

Table 3 The calculated velocity and shear stress in the tested syringe (1ml I.D.: 4.78mm, 2ml I.D.: 8.66) and needles (19G, 27G) and the

pressure drop (Ps - Pn) from syringe to needle (Ps: the pressure in the liquid near the piston in the syringe, Pn: the pressure in the liquid at the

outlet of the needle)

Injection

rate

(ml/min)

Syringe

volume

(ml)

Syringe

ID (mm)

Mean

velocity in

syringe

(mm/s)

Needle

Gauges

Needle ID

(mm)

Mean

velocity in

needle

(mm/s)

Pressure

drop

Ps-Pn (Pa)

Shear stress

in syringe

(mPa)

Shear stress

in needle

(Pa)

0.85

1 4.78 0.79

19G 0.686 38.35 0.73 1.32 0.45

27G 0.21 409.22 83.73 1.32 15.59

3

2 8.66

0.85

19G 0.686 135.35 9.16 0.78 1.58

27G 0.21 1444.31 1043.02 0.78 55.02

17

3.2 The variation in diameter, concentration and acoustic characterisation

In the following figures the headings of the groups are such that ‘Needle Gauge’

group indicates that only the needle gauge changed, ‘Injection Rate’ group indicates

that only the injection rate changed and ‘Temperature’ group indicates that only

temperature changed with respect to the in vitro data. For the cases of in vivo data, all

three parameters changed.

Figure 1 shows the mean diameter of Definity, SonoVue and MicroMarker as a

function of different injection regimes. The mean diameters in in vitro case are larger

than the values of Definity and SonoVue shown in Table 1 as the mean diameter in

this study was from the volume-based size measurement by Mastersizer, while the

values from the manufacturer use the number-based size measurement. Compared

with the in vitro case, the mean diameter of Definity decreases with increasing needle

gauge, decreasing injection rate and increasing temperature. The individual impact of

needle gauge makes no significant difference in the mean diameter of SonoVue, but

lower injection rate, and the combined effect of higher temperature and lower

injection rate show a significant decrease in the mean diameter of SonoVue.

MicroMarker exhibits the smallest mean diameter of the three UCAs. Unlike the mean

diameters of Definity and SonoVue, MicroMarker shows no significant variation

between in vitro and in vivo experimental regimes.

18

Fig.1: Volume-based mean diameter of Definity, SonoVue and MicroMarker under

different experimental regimes. Needle gauge group indicating that only the needle

gauge changed, injection rate group indicating that only the injection rate changed and

temperature group indicating that only temperature changed with respect to the in

vitro data. In vivo data, all three parameters changed as detailed in text. Significance

determined between ‘in vitro’ data set and other experimental regimes so that no mark

indicates no significant difference, **P<0.01, ***P<0.001

The concentration of each of the three UCAs significantly decreases with increasing

needle gauge in Figure 2. Only SonoVue presents a significant increase in

concentration with decreasing injection rate. Both SonoVue and Definity show a

decrease in concentration with temperature. In a comparison between in vivo and in

vitro applications, MicroMarker concentration displays no significant variation but

both SonoVue and Definity display a reduction in concentration.

19

Fig.2: Concentration of Definity, SonoVue and MicroMarker under different

experimental regimes, * P<0.05, **P<0.01, ***P<0.001. Group headings similar to

that displayed in Figure 1

In Figure 3 the attenuation of ultrasound through the suspensions of Definity,

SonoVue and MicroMarker in the frequency range 17-31 MHz is presented. No

significant variation in the attenuation of Definity was found with changing needle

gauge and injection rate. Compared to the in vitro data SonoVue exhibited significant

decreases in attenuation with decreasing injection rate, increasing needle gauge and

temperature. Conversely, for MicroMarker, the attenuation significantly increases

from RT to BT, but neither increasing needle gauge nor injection rate makes a

significant difference to attenuation.

20

Fig.3: The attenuation comparison of Definity, SonoVue and MicroMarker, No mark:

no significant difference, * P<0.05, **P<0.01, ***P<0.001. Group headings similar to

that displayed in Figure 1.

From Figure 4, increasing gauge size needle only shows a significant, but small,

impact on the CTR of SonoVue. Using a lower injection rate decreases the CTR of

Definity and SonoVue significantly but increases the CTR of MicroMarker. The

variation in temperature increases the CTR of SonoVue and MicroMarker. Overall a

change from in vitro to in vivo conditions results in a small but significant decrease in

CTR for SonoVue and a highly significant increase for MicroMarker.

21

Fig.4: The CTR comparison of Definity, SonoVue and MicroMarker, no mark: no

significant difference, ** P<0.01, ***P<0.001. Group headings similar to that

displayed in Figure 1

The NSI of Definity, SonoVue and MicroMarker are shown in Figure 5. For Definity,

NSI shows no significant variation with needle gauge and injection rate. Significant

decrease in NSI of SonoVue was found with increase in temperature. The variation in

the trend of SonoVue is consistent with the variation of MicroMarker in the

temperature and in vivo cases. MicroMarker had the largest NSI of the three agents.

22

Fig.5: The normalized subharmonic intensity comparison of Definity, SonoVue and

MicroMarker, No mark: no significant difference, **P<0.01, ***P<0.001. Group

headings similar to that displayed in Figure 1

4 DISCUSSION

4.1 The impact of needle gauge on the mean diameter, concentration and acoustic

properties of the MBs at RT

The concentration of Definity, SonoVue and MicroMarker obtained from the study of

needle gauge size between 19G and 27G at RT agrees with Talu’s study [20] which

show that increasing needle gauges (i.e., narrower ID) reduces the concentration of

the MBs because of the aggravated diffusion of the MBs due to an increment in the

forced compression named as shear stress (Table 3). The concentration of SonoVue

was found to decrease by far the most in the three UCAs. This may also be the reason

for the decrease in mean diameter of Definity agreeing with previous studies [20, 21].

23

However, no significant difference was found in the mean diameter for SonoVue MB.

Definity, SonoVue and MicroMarker are poly-disperse MBs of concentration

1.2×1010MBs/ml [10], 2-5×108 MBs/ml [24] and 2×109 MBs/ ml [26] respectively.

Talu et al. [20] showed less than 10% variation in mean diameter of in-house MBs

when using 27G needle at an injection rate of 0.1 ml/sec (3ml/min = 0.05 ml/sec).

Although the structure and the size distribution of SonoVue MBs are comparable to

the in-house MBs described in the Barrack et al study, they are different from the

small MBs (0.95 ± 0.75 µm) with a narrow distribution in Talu et al. [20] study.

However the concentration of MBs used in this study is similar to Talu’s study

(0.1/0.5/1×109 MBs/ml) and approximately a factor of 500 greater than that used in the

Barrack’s study. Therefore it is likely that the differences between the tested agents

are caused by differences in concentration and size distribution.

For Definity in a frequency range of 17-31MHz and at RT, only the measured NSI

was found to decrease with increasing gauge needle. However a significant decrease

was found in the attenuation and CTR of SonoVue at RT with increasing needle

gauge that may be due to the reduction in mean diameter and concentration of MBs.

4.2 The impact of injection rate on the mean diameter, concentration and acoustic

properties of the MBs

The reduction in injection rate from 3 ml/min to 0.85 ml/min at RT is found to

decrease the mean diameter of both SonoVue and Definity MBs. This is not in

agreement with the trend from previously reported studies [20, 21]. However in our

study, in addition to the difference in the structure, concentration and size distribution

24

between the tested MBs, from Table 3 it can be seen that at RT the decrease in mean

diameter from high to low injection rate may be attributed to a lower shear stress

(0.78 mPa) within the 2ml syringe used for high injection rates compared to a higher

shear stress (1.32mPa) experienced within the 1ml syringe used for low injections

rates. The results in Talu’s and Barrack’s studies support these observations as the

change in mean diameter of MBs in Talu’s study is less than 5% using a 23G needle

[20]; while in this study using a 19G needle (larger I.D.) a smaller reduction in the

variation in mean diameter would be expected. The syringe was placed horizontally

on the syringe pump during injection so it is important to note that at a low injection

rate there was sufficient time for large MBs to float towards the wall of the syringe

and therefore be exposed to the increasingly higher shear stress [21, 22, 30]. However,

the total time of the needle being held horizontally was a maximum of 1 minute from

set-up to the UCA being output into an Eppendorf. From calculations based upon

Goertz et al [23], it can be shown that even a bubble of as large a diameter of 5

micron requires approximately 3 minutes to float from the center of the syringe to the

syringe wall, which is significantly longer than the limited time of bubble passage

through the syringe. Unlike Definity, MicroMarker showed no significant variation in

concentration with changing injection rate. However, the concentration of

microbubbles decreases for SonoVue with increasing injection rate similar to the

results in Barrack’s study [21].

For Definity and SonoVue in the frequency range of 17-31 MHz the measured CTR

was found to decrease significantly with decreasing injection rate with respect to the

in vitro data. From Table 3, using 19G needle, the pressure drop and the shear stress

in the needle at an injection rate of 3 ml/min is 10 times and 5 times greater

25

respectively than the corresponding values at 0.85 ml/min. Additionally, as analysed

in section 4.2 that the mean diameter of Definity and SonoVue decreased at lower

injection rate, i.e., large MBs are more likely to be disrupted at a higher injection rate

than at a lower injection rate. This may explain the significant reduction in CTR

observed for SonoVue and Definity at lower injection rates. Conversely a significant

increase in CTR with injection rate was observed for MicroMarker. The mean

diameter of MicroMarker was smaller (in vitro case in Figure 1) than that of Definity

and SonoVue, thus the floatation of large MBs of MicroMarker towards the wall of

syringe during the slow injection process is likely to have less impact on the size

distribution and concentration for Definity and SonoVue.

4.3 The impact of temperature on the mean diameter, concentration and acoustic

properties of the MBs

The mean diameters of SonoVue MBs was shown to increase from RT to BT which

agreed with previous results [16]. The process of MBs dissolution was previously

found to be in 3 phases: (1) an initially quick growth, (2) steady dissolution [31], (3)

phase changes from low vapour pressure to vapour-to-liquid under Laplace pressure

[32, 33]. In the first growth stage, it was proposed that the increase in temperature

enabled the expansion of SonoVue MBs by weakening the chemical bonds between

lipid molecules and changing the shell elasticity and surface tension [16]. However in

our studies, Definity showed a significant decrease in mean diameter form RT to BT.

Because of the different shell components of the two UCAs, when close to the phase

transition temperature, Definity may tend to dissolve faster than SonoVue and lead to

26

the overall reduction in mean diameter. The concentration of both Definity and

SonoVue decreases significantly at BT.

The results from this study show that the attenuation of SonoVue reduces from RT to

BT in the frequency range 17-31MHz. Previously published data of SonoVue

measured at 3.5 MHz and at 100kPa peak negative pressure found attenuation

increased with temperature below 40oC [19]. However, the frequency of insonation in

this study is much greater than the reported resonance frequency range of SonoVue

MBs (1-3 MHz) [25] and we have shown that the concentration of SonoVue MBs

reduces significantly at BT. In comparison, in our study the attenuation of

MicroMarker was found to increase significantly with temperature suggesting that at

higher temperatures there may be an increase in mean diameter of the MB population

giving a larger attenuation. Previous studies at RT have shown that MicroMarker

demonstrated an increase in attenuation up to 21MHz [34]. In addition the higher

values of attenuation exhibited by MicroMarker in BT measurements compared to RT

measurements in our studies are consistent with similar measurements made by

Raymond et al [14] at lower frequencies. The variation of acoustical properties for

Definity as a function of temperature is not included in this paper.

The CTRs of SonoVue and MicroMarker were found to significantly increase at BT

compared with RT. For SonoVue, although the number of microbubbles decreased at

BT, there was an increase in mean diameter. The increased CTR may be caused by the

larger scattering cross-section that can be attributed to an increase in mean diameter.

The NSIs of SonoVue and MicroMarker decrease from RT to BT. de Jong et al (2000)

has shown that maximal subharmonics are generated by microbubbles resonating at

27

half of the driving frequency [35], which is fixed at 15MHz in this study. As we have

shown in Figure 1 that the diameters of SonoVue and MicroMarker bubbles increase

with temperature they will therefore resonate at lower frequencies potentially outwith

the frequency bandwidth of the transducer used in this study.

4.4 The combined effect of needle gauge, injection rate and temperature and future

research

Based on the above discussions of the individual impact of the three factors (needle

gauge, injection rate and temperature), no clear trend is evident in the changes in the

mean diameter, concentration and acoustic parameters of the three UCAs. However,

for SonoVue and MicroMarker, significant variation was found in both size and

acoustic properties when needle gauge, temperature and injection rate were changed

from a typical in vitro to a typical in vivo experimental design. The differences

between the in vivo and in vitro settings are the cumulative result of variations in the

parameters with one or more factors predominating and effectively determining the

measured outcome. In the future, studying the variation in single MB of different

diameters at distinct environments might be of interest to give a quantitative answer to

this question. Details of this variation will allow researchers to estimate the response

of MBs under different environments and explain the discrepancy between in vitro

and in vivo experiments.

5 CONCLUSION

In this paper, the influence of needle gauge and injection rate on the mean diameter,

concentration and acoustic properties of Definity, SonoVue and MicroMarker over the

28

frequency range of 17-31MHz and the influence of temperature on SonoVue and

MicroMarker over the same frequency range were investigated. In particular, the

influence of these parameters on typical in vitro settings (19G needle, 3ml/min at RT)

and in vivo settings (27G needle, 0.85ml/min at BT) were compared. Although no

consistent trend was found for each individual experimental set-up change, we found

that the mean volume-based diameter and concentration of both SonoVue and

Definity reduced significantly with a tenfold reduction in concentration when

measured under typical in vivo experimental conditions compared to in vitro

conditions. For MicroMarker, neither the mean volume-based diameter nor

concentration changed significantly. With respect to the acoustic properties of the

UCAs we found that the attenuation, CTR and NSI of SonoVue significantly

decreased when moving from typical in vitro to in vivo experimental set-ups. From

our limited measurements of Definity, we found no significant change in attenuation,

CTR and NSI with needle gauge. For MicroMarker we found that, while the

attenuation and CTR increased, the NSI decreased when changing from typical in

vitro to in vivo experimental set-ups. These results suggest that in similarity to results

obtained at lower frequencies, care must be taken in translating results obtained in

vitro to preclinical in vivo imaging studies. Moreover based on these results, it is

likely that other variables such as choice of diluent (saline or blood) and the gas

saturation level of the testing environment may have significant effect on the acoustic

measurements and thus are worthy of further exploration at high frequencies.

ACKNOWLEDGEMENT

29

Dr. Sun is funded by China Scholarship Council/University of Edinburgh joint

scholarship and acknowledges the funding from National Natural Science Foundation

of China (81501472).

CONFLICT OF INTEREST

Dr. Moran acknowledges receipt of a small research grant from Lantheus Medical

Imaging.

REFERENCE

[1] F.S. Foster, J. Hossack, S.L. Adamson, Micro-ultrasound for preclinical imaging,

Interface Focus, 1 (2011) 576-601.

[2] D.E. Goertz, E. Cherin, A. Needles, R. Karshafian, A.S. Brown, P.N. Burns, F.S.

Foster, High frequency nonlinear B-scan imaging of microbubble contrast agents,

Ultrasonics, Ferroelectrics and Frequency Control, IEEE Transactions on, 52 (2005)

65-79.

[3] B.L. Helfield, E. Cherin, F.S. Foster, D.E. Goertz, Investigating the Subharmonic

Response of Individual Phospholipid Encapsulated Microbubbles at High Frequencies:

A Comparative Study of Five Agents, Ultrasound Med. Biol., 38 (2012) 846-863.

[4] M.R. Sprague, E. Chérin, D.E. Goertz, F.S. Foster, Nonlinear Emission from

Individual Bound Microbubbles at High Frequencies, Ultrasound Med. Biol., 36

(2010) 313-324.

30

[5] S. Stapleton, H. Goodman, Y.-Q. Zhou, E. Cherin, R.M. Henkelman, P.N. Burns,

F.S. Foster, Acoustic and Kinetic Behaviour of Definity in Mice Exposed to High

Frequency Ultrasound, Ultrasound Med. Biol., 35 (2009) 296-307.

[6] D.L. Miller, C. Dou, R.C. Wiggins, B.L. Wharram, M. Goyal, A.R. Williams, An

in vivo rat model simulating imaging of human kidney by diagnostic ultrasound with

gas-body contrast agent, Ultrasound Med. Biol., 33 (2007) 129-135.

[7] C.M. Howard, F. Forsberg, C. Minimo, J.B. Liu, D.A. Merton, P.P. Claudio,

Ultrasound guided site specific gene delivery system using adenoviral vectors and

commercial ultrasound contrast agents, J. Cell. Physiol., 209 (2006) 413-421.

[8] D.A. Heaton, S. Golding, C.P. Bradley, T.A. Dawson, S. Cai, K.M. Channon, D.J.

Paterson, Targeted nNOS gene transfer into the cardiac vagus rapidly increases

parasympathetic function in the pig, J. Mol. Cell. Cardiol., 39 (2005) 159-164.

[9] E.R. Wisner, K. Ferrara, J.D. Gabe, D. Patel, T.G. Nyland, R.E. Short, T.B.

Ottoboni, Contrast enhanced intermittent power Doppler ultrasound with sub-micron

bubbles for sentinel node detection, Acad. Radiol., 9 (2002) S389-S391.

[10] Lantheus Medical Imaging, Prescribing Information of Definity vial for

(perflutren lipid microsphere) injectable suspension, in, Lantheus Medical Imaging

Inc, 2011.

[11] G. Pu, M.A. Borden, M.L. Longo, Collapse and Shedding Transitions in Binary

Lipid Monolayers Coating Microbubbles, Langmuir, 22 (2006) 2993-2999.

[12] J.M. Zook, W.N. Vreeland, Effects of temperature, acyl chain length, and flow-

rate ratio on liposome formation and size in a microfluidic hydrodynamic focusing

device, Soft Matter, 6 (2010) 1352-1360.

[13] H.C. Berg, Random walks in biology, Princeton Univ Pr, 1993.

31

[14] J.L. Raymond, K.J. Haworth, K.B. Bader, K. Radhakrishnan, J.K. Griffin, S.-L.

Huang, D.D. McPherson, C.K. Holland, Broadband Attenuation Measurements of

Phospholipid-Shelled Ultrasound Contrast Agents, Ultrasound Med. Biol., 40 (2014)

410-421.

[15] C. Greis, Technology overview: SonoVue (Bracco, Milan), European Radiology

Supplements, 14 (2004) P11-P15.

[16] H. Mulvana, E. Stride, M. Tang, J.V. Hajnal, R. Eckersley, Temperature-

Dependent Differences in the Nonlinear Acoustic Behavior of Ultrasound Contrast

Agents Revealed by High-Speed Imaging and Bulk Acoustics, Ultrasound Med. Biol.,

37 (2011) 1509-1517.

[17] C. Guiot, G. Pastore, M. Napoleone, P. Gabriele, M. Trotta, R. Cavalli, Thermal

response of contrast agent microbubbles: Preliminary results from physico-chemical

and US-imaging characterization, Ultrasonics, 44, Supple (2006) e127-e130.

[18] H.J. Vos, M. Emmer, N. de Jong, Oscillation of single microbubbles at room

versus body temperature, in: Ultrasonics Symposium, 2008. IUS 2008. IEEE, 2008,

pp. 982-984.

[19] H. Mulvana, E. Stride, J.V. Hajnal, R.J. Eckersley, Temperature Dependent

Behavior of Ultrasound Contrast Agents, Ultrasound Med. Biol., 36 (2010) 925-934.

[20] E. Talu, R.L. Powell, M.L. Longo, P.A. Dayton, Needle Size and Injection Rate

Impact Microbubble Contrast Agent Population, Ultrasound Med. Biol., 34 (2008)

1182-1185.

[21] T. Barrack, E. Stride, Microbubble Destruction During Intravenous

Administration: A Preliminary Study, Ultrasound Med. Biol., 35 (2009) 515-522.

32

[22] R.J. Browning, H. Mulvana, M. Tang, J.V. Hajnal, D.J. Wells, R.J. Eckersley,

Influence of Needle Gauge On In Vivo Ultrasound and Microbubble-Mediated Gene

Transfection, Ultrasound Med. Biol., 37 (2011) 1531-1537.

[23] D.E. Goertz, N. de Jong, A.F.W. van der Steen, Attenuation and Size

Distribution Measurements of Definity(TM) and Manipulated Definity(TM)

Populations, Ultrasound Med. Biol., 33 (2007) 1376-1388.

[24] M. Schneider, SonoVue, a new ultrasound contrast agent, European Radiology, 9

(1999) S347-S348.

[25] J. Gorce, M. Arditi, M. Schneider, Influence of Bubble Size Distribution on the

Echogenicity of Ultrasound Contrast Agents: A Study of SonoVue (TM), Invest.

Radiol., 35 (2000) 661-661.

[26] Visualsonics, Vevo MicroMarker™ Non-Targeted Contrast Agent Kit:

Instructions and Protocols Rev1.4, (2012).

[27] H. Mulvana, E. Stride, M.-X. Tang, J.V. Hajnal, R.J. Eckersley, The Influence of

Gas Saturation on Microbubble Stability, Ultrasound Med. Biol., 38 (2012) 1097-

1100.

[28] C. Sun, S.D. Pye, J.E. Browne, A. Janeczko, B. Ellis, M.B. Butler, V. Sboros,

A.J.W. Thomson, M.P. Brewin, C.H. Earnshaw, C.M. Moran, The Speed of Sound

and Attenuation of an IEC Agar-Based Tissue-Mimicking Material for High

Frequency Ultrasound Applications, Ultrasound Med. Biol., 38 (2012) 1262-1270.

[29] C. Sun, V. Sboros, M.B. Butler, C.M. Moran, In Vitro Acoustic Characterization

of Three Phospholipid Ultrasound Contrast Agents from 12 to 43 MHz, Ultrasound

Med. Biol., 40 (2014) 541-550.

33

[30] M. Kaya, T.S. Gregory V, P.A. Dayton, Changes in Lipid-Encapsulated

Microbubble Population During Continuous Infusion and Methods to Maintain

Consistency, Ultrasound Med. Biol., 35 (2009) 1748-1755.

[31] H.D. Van Liew, M.E. Burkard, Behavior of bubbles of slowly permeating gas

used for ultrasonic imaging contrast, Invest. Radiol., 30 (1995) 315-315.

[32] A. Kabalnov, D. Klein, T. Pelura, E. Schutt, J. Weers, Dissolution of

multicomponent microbubbles in the bloodstream: 1. theory, Ultrasound Med. Biol.,

24 (1998) 739-749.

[33] J.J. Kwan, M.A. Borden, Microbubble Dissolution in a Multigas Environment,

Langmuir, 26 (2010) 6542-6548.

[34] E. Huo, B. Helfield, D. Goertz, Scaling of viscoelastic shell properties of lipid

encapsulated microbubbles with frequency, The Journal of the Acoustical Society of

America, 128 (2010) 2280-2280.

[35] N. de Jong, P.J.A. Frinking, A. Bouakaz, F.J. Ten Cate, Detection procedures of

ultrasound contrast agents, Ultrasonics, 38 (2000) 87-92.

34

Research highlights

� When changing from typical in vitro to in vivo experimental conditions, Definity

and SonoVue demonstrated a significant reduction in mean diameter.

MicroMarker did not demonstrate any significant change in diameter.

� When changing from typical in vitro to in vivo experimental conditions, Definity

and SonoVue demonstrated a significant tenfold reduction in concentration.

Micromarker did not demonstrate any significant change in concentration.

� When changing from typical in vitro to in vivo experimental conditions, SonoVue

demonstrated a significant reduction in attenuation, contrast to tissue ratio (CTR)

and normalized subharmonic intensity (NSI).

� When changing from typical in vitro to in vivo experimental conditions,

MicroMarker demonstrated a significant increase in attenuation, CTR but a

reduction in NSI.

� Care must be taken in translating results obtained in vitro to preclinical imaging

studies.